CN107050010A - Application of the Cichoric acid in the medicine of anti respiratory syncytial virus is prepared - Google Patents

Abstract

The invention discloses the new application of Cichoric acid --- the application in the medicine of anti respiratory syncytial virus is prepared.The present inventor screens using Chemistry for Chinese Traditional Medicine method to the antiviral ingredients in plant Echinacea, and optimal antiviral activity composition is found that by extracorporeal antivirus effect effect experiment --- Cichoric acid, and find that Cichoric acid is most strong to the Anti-viral activity in vitro of Respiratory Syncytial Virus(RSV) (RSV), better than comparison medicine Ribavirin, therefore, the medicine for preparing anti respiratory syncytial virus can be used for by active component of Cichoric acid, during concrete application, acceptable matrix or auxiliary material in pharmacy can be added in right amount, can also be with appropriate Drug combination.

Description

Application of the Cichoric acid in the medicine of anti respiratory syncytial virus is prepared

Technical field

The present invention relates to application of the Cichoric acid in the medicine of anti respiratory syncytial virus is prepared.

Background technology

Echinacea also known as Echinacea, purple coneflower, are herbaceos perennial, composite family Echinacea.Eighties of last century 70 years Generation, Echinacea introduces China, is cultivated as ornamental plant；The nineties, Xiao Peigen academician first adjusts Echinacea as immune Section agent makes referrals to the country, and domestic various regions start to introduce a fine variety as medicinal plant, and gradually puts into GAP standardized productions, so that Ensure the materials safety problem of research and development.The existing several Echinacea preparations of recent year are developed and are applied to face Bed, such as " golden screen capsule " (Zhejiang Zhengda Qingchunbao Pharmaceutical Co., Ltd), " purple sealwort " (limited public affairs of Chengdu Diao Pharmaceutical Group Department) etc., it is the compound preparation of Echinacea and the qi-invigorating herb Radix Astragali.

Widely, main application is improved immunity of organism level, anti-inflammatory, scavenging activated oxygen with anti-for the application of Echinacea Oxidation, antitumor, antiviral etc..Foreign countries are in the research of Echinacea antivirus action, Wacker is trained using mouse L-929 cells Foster method confirms that Echinacea Purpurea Herb P.E can indirectly suppress the activity of influenza virus, herpesviral and vesicular stomatitis virus； May has found that the water extract of Echinacea purpurea can substantially suppress influenza virus, herpesviral and poliovirus activity.At present, The domestic research to Echinacea antivirus action is also in the starting stage, and document report focuses mostly in animal husbandry.Feng Shanxiang is investigated Protective effect of the Echinacea compound to infection newcastle disease virus chick, artificial challenge infectious bursa of Fabricius virus chick, And point out that Echinacea compound can be used for newcastle disease, the prevention and treatment of infections chicken cloacal bursa.On a cellular level, it is purple Cone chrysanthemum compound preparation has obvious inhibitory action to NDV, and effect is better than Echinacea.

In the antiviral study of Echinacea, its extract infected by influenza, Coxsackie virus B, blister sore is had proven to Cytopathy caused by poison, HSV- II has different degrees of inhibitory action, to Ard₃、Ard₇, parainfluenza virus, ECHO₁₁And The experiment in vitro of the grades of HSV- I is then without positive effect.Current China is also confined to stimulate to the research for introducing a fine variety Echinacea pharmacological activity In terms of immune level, people is with anti-virus formulation not yet full-scale development.Echinacea is used for controlling for human viral disease realizing During treatment, the problems such as still having unknown medicinal material shortage of standard, antiviral activity composition, antivirus action mechanism and unclear target spot Lie across at the moment.Therefore it is that it obtains full-scale development in China to carry out total quality evaluation study and pharmacology activity research to Echinacea The premise of application.

Echinacea is used as a kind of good Immune enhancement herbal medicine, its chemical composition and the existing more report of Ingredients Active.Mesh It is preceding in the chemical composition and structural research of genus echinacea plant and its preparation, it is separated go out Caffeic acids derivative, alkyl acyl Amine, polysaccharide, volatile oil, alkaloid, flavones, polyacetylene and other class chemical compositions etc..Cichoric acid is pole in Echinacea For one of important immune active ingredient, pharmacological research in recent years shows, there is Cichoric acid enhancing immunologic function and anti-inflammatory to make With, and hyaluronidase can be suppressed, protect the influence of free radical of the collagen from degraded can be caused.In the prior art not yet See the relevant report in terms of anti respiratory syncytial virus on Cichoric acid.

The content of the invention

For above-mentioned prior art, the present invention is through research, it was found that the new application of Cichoric acid --- preparing preventing respiratory Application in the medicine of syncytial virus.

The present invention is achieved by the following technical solutions：

The present inventor screens using Chemistry for Chinese Traditional Medicine method to the antiviral ingredients in plant Echinacea, and leads to Cross extracorporeal antivirus effect effect experiment and be found that optimal antiviral activity composition --- Cichoric acid, and find that Cichoric acid is closed to respiratory tract The Anti-viral activity in vitro of cellular virus (RSV) is most strong, better than comparison medicine Ribavirin, therefore, it can using Cichoric acid as activity into Point be used to prepare the medicine of anti respiratory syncytial virus, during concrete application, can add in right amount in pharmacy acceptable matrix or Auxiliary material, can also be with appropriate Drug combination.

Echinacea pharmaceutical active complicated component, have Caffeic acids derivative, alkylamide, polysaccharide, volatile oil, Alkaloid, flavones, polyacetylene and other class chemical compositions etc., the present invention are not only found that Echinacea Purpurea Herb P.E to respiratory syncystial Virus has inhibitory action, it is also expressly that Cichoric acid is the active ingredient of external anti respiratory syncytial virus, to the disease-resistant of Echinacea Malicious research, the further research of Echinacea prescribed preparation etc. are significant.

Brief description of the drawings

Fig. 1：：Normal cell and viral sick cell, wherein, A：Normal VERO cells (4 × 40)；B：The virosis of HSV- I Become (4 × 10)；C：Normal VERO cells (4 × 10)；D：RSV virus lesions (4 × 10)；E：Normal RD cells (4 × 10)；F： EV71 virus lesions (4 × 10).

Fig. 2：The selection result block diagram of Echinacea extracorporeal antivirus effect spectrum.

Fig. 3：Echinacea modifiedly extracted method result block diagram.

Fig. 4：The antiviral result block diagram of Echinacea water extract D101 type macroreticular resin eluates.

Fig. 5：D101 macroreticular resin post separation sample Ps AD monitoring figures.

Fig. 6：D101 macroreticular resin post separation active component liquid chromatograms.

Fig. 7：Quick prepare compound efficient liquid phase detection chromatogram.

Fig. 8：Cichoric acid reference substance cleaved fragment information.

Fig. 9：Prepare sterling compound cleaved fragment information.

Figure 10：Echinacea prepares sterling Antiviral breeding result block diagram.

Embodiment

With reference to embodiment, the present invention is further illustrated.

Involved instrument, reagent, material etc. in following embodiments, are existing in the prior art unless otherwise noted Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Involved experimental method in following embodiments, inspection Survey method etc., is existing normal experiment method in the prior art, detection method etc. unless otherwise noted.

Test the screening of Echinacea extracorporeal antivirus effect spectrum

1. laboratory apparatus and material

1.1 medicine Echinacea dry aerial parts medicinal materials are purchased from Anhui Province Tongcheng City Wei Qing medicinal plants Co., Ltd, powder Broken machine is crushed, standby；(Qilu Pharmaceutical Co., Ltd., specification is 50mg/mL, lot number to ribavirin injection：1501166141).

(VERO is HSV- I, RSV sensitive cells strain, by Shandong Province's medical science to 1.2 cell African green monkey kidney cells Microbiological Lab of institute preserves)；(the sensitive cells strain that RD is EV71, by the micro- life of Shandong Academy of Medical Sciences of rhabdomyoma cell Thing laboratory is preserved).

1.3 viral seed culture of viruses HSV- I：F plants, quoted from microbiology teaching and research room of Shandong Medical University, cured by Shandong Province within 1996 Microbiological Lab of subject institute preserves；RSV：Long plants, in October, 2000 is quoted from Virology Inst., China Academy of Preventive Medicine Sciences's poison Room is planted, this section office preserves；EV71, is isolated from Jinan Children's Hospital ICU wards and dies of illness infant (Genbank registration numbers： HQ825317)。

1.4 reagent n-butanols, petroleum ether, ethyl acetate, acetone, ethanol, dimethyl sulfoxide (DMSO) (Tianjin richness space fine chemistry industry Co., Ltd's product, is that analysis is pure)；Cell culture fluid (1640 cell culture fluids, GIBCO products, containing 11% NBCS And 100U/mL penicillin and streptomysin, put 4 DEG C it is standby)；Cell maintenance medium (1640 cell culture fluids, GIBCO products, containing 2% NBCS and 100U/mL penicillin and streptomysin)；PBS (pH7.2,8g containing NaCl, KCl 0.2g, KH₂PO₄0.2g、 Na₂HPO₄2.9g, plus distilled water, to 1000mL, filtration sterilization, 4 DEG C of separating device is standby)；Cell dissociation buffer (0.25% pancreatin, Macgene Products, dispense to penicillin bottle, put -20 DEG C of refrigerators standby).

1.5 instrument CO₂Constant incubator (SANYO GS Products)；(Jinan Xin Beixi companies produce Biohazard Safety Equipment Product)；Superclean bench (Jinan Long Hong cleaning equipments Co., Ltd)；High-pressure sterilizing pot (Shanghai Bo Xun Industrial Co., Ltd.s medical treatment Instrument factory)；Double two-sided clean work station (Suzhou purifying apparatus factory)；Inverted microscope (Olympus Products)；It is desk-top from Dynamic balancing centrifuge (Changsha Xiang Zhi centrifuges Co., Ltd)；Centrifuge (congratulates the desk-top refrigerated centrifuges of Li Shi)；Ultra low temperature freezer (- 80 DEG C, 725 types, ThermoForma companies)；Low temperature refrigerator (- 40 DEG C, SANYO GS company；- 20 DEG C, the U.S. water chestnut of middle section is public Take charge of product；4 DEG C, brother company of Haier product)；Liquid nitrogen container (Sichuan liquid nitrogen tank farms product)；96 porocyte culture plates (the U.S. GIBCO Products)；Degerming syringe needle filter (Solution of U.S. Pall Corporation, aperture：0.22 μm, diameter 13mm)；Pipettor (German Eppendorf products, model：20-200μl；100-1000μl)；Suction pipette head (Germany Eppendorf products, supporting with pipettor)；Sonic oscillation cleaning device (Kunshan Ultrasonic Instruments Co., Ltd.)；The omnipotent powder of high speed Broken machine (Beijing forever bright Medical Instruments Co., Ltd, FW-100)；Water-bath (establishing instrument and meter Co., Ltd in Shanghai) electricity Hot blast drying box (Tianjin Lai Boterui experimental instruments and equipment limiteds, GFL-230).

2. experimental method

2.1 prepare opposed polarity solvent Echinacea Purpurea Herb P.E

Echinacea medicinal powder 40g is taken, the distilled water for adding 8 times of amounts carries out decoction 2h, suction filtration；The dregs of a decoction after filtering are taken to add together Sample amount distilled water continues to decoct 2h, suction filtration；Filtrate it will merge twice, water-bath is volatilized.Change respectively 80% ethanol, 100% ethanol, Petroleum ether, ethyl acetate, n-butanol repeat above-mentioned processing step, prepare Echinacea opposed polarity extractive part.Water extract with Cell maintenance medium is dissolved, and other extracts are dissolved with DMSO, and 0.22 μm of filter filtration sterilization is standby.

Take Echinacea powder 40g, plus 8 times of amounts 50% acetone ultrasonic extraction 1h, suction filtration, filter residue adds 8 times of amounts 50% the third again Ketone repeats ultrasound 1h, suction filtration；Filtrate merges twice, and water-bath is volatilized, and dissolves appropriate with DMSO, 0.22 μm of filter filtration sterilization is standby With.

2.2 opposed polarity solvent Echinacea Purpurea Herb P.E antiviral study in vitro

2.2.1 the recovery and passage of cell

Vero, RD cell frozen is taken out from liquid nitrogen, is invested in 37 DEG C of warm water and rocks rapidly, it is rapid in 1min Thaw.800rpm centrifuges 5min, and cell is suspended, is transferred to containing cell culture fluid by abandoning supernatant using cell culture fluid Tissue Culture Flask in, 37 DEG C, 5%CO₂Static gas wave refrigerator in incubator.Cell grows up to individual layer after 2d, and 0.25% pancreatin digests simultaneously 1:2 passages, can be used for testing when growing up to individual layer to cell.

2.2.2 the amplification of virus

The above-mentioned long cell to individual layer is taken, abandoning supernatant will deposit in HSV- I, RSV, EV71 under the conditions of -80 DEG C Virus is inoculated on corresponding sensitive cells respectively after thawing.In 35 DEG C, 5%CO₂Under the conditions of incubator in be incubated 1h, take out Add after cell maintenance medium, continue to place and cultivated in incubator.Day by day Microscopic observation cytopathy (cytopathic Effect, CPE), when CPE reaches 90%, in ultra low temperature freezer multigelation 3 times, piping and druming centrifugation (20000r/min, 15min), Take supernatant to dispense, carry out mark, -40 DEG C of refrigerators freeze standby.

2.2.3 the measure of virus virulence

Herpes simplex virus Ⅰ liquid, Respiratory Syncytial Virus(RSV) liquid are done 10 multiple proportions and is serially diluted with cell maintenance medium, laterally Be seeded in 96 orifice plates and grow up on the vero cells of individual layer successively, longitudinal direction repeats 3 holes, while set normal cell controls, put 35 DEG C, Cultivated in 5%CO2 incubators；Take EV71 virus liquids to do after the dilution of ten multiple proportions systems with same method, plant thin in the RD for growing up to individual layer On born of the same parents.Observed after 48h under inverted microscope, lesion occurs in cell, be considered as viral virulence, terminated culture, observe and record cell Lesion rate.

With Reed-Muench Liang Shi methods, i.e. formula (1), (2), each viral half cell infection amount TCID is calculated₅₀ (50%tissue culture infective dose).

Formula (2) TCID50=Anti log (logC+pd × logCm)

Wherein：Pd=cells ratio away from；P1=is higher than 50% lesion percentage；P2=is less than 50% lesion percentage；C=is high In the dilution factor of 50% lesion rate；Cm=doubling dilution coefficients.

2.2.4 the measure of drug cytotoxicity

Take under 2.1 standby opposed polarity extract appropriate, 2 multiple proportions made of cell maintenance medium and are serially diluted, totally 12 it is dilute Degree of releasing, is loaded onto in 96 orifice plates and has grown up on vero, RD cell of individual layer respectively, each dilution factor longitudinal direction 3 holes of repetition, 37 DEG C, Cultivated in 5%CO2 incubators.Micro- Microscopic observation lesion situation after 24 hours, treats that virus control lesion rate reaches 90%, termination Culture, observes and records cytopathy (CPE) percentage of every kind of extract, and medicine half is calculated using Reed-Muench methods Number poisoning concentration (TC₅₀), and determine maximal non-toxic concentration (TC₀)。

2.2.5 the influence that medicine is acted on pathological changes caused by virus

Opposed polarity extract standby under 2.1 is taken to make 2 multiple proportions series, dilution totally 12 dilutions of cell maintenance medium Degree, is loaded onto on individual layer RD, VERO cell in 96 orifice plates successively by 100 μ L/ holes respectively, and each dilution factor repeats 3 holes, if sharp Ba Weilin parenteral solutions stoste is positive control, and sets cell controls, virus control.Except cell controls plus 100 μ L cell maintenance mediums Outside, each hole adds measures TCID under 2.2.3₅₀The μ L of virus liquid 100 of concentration.Put 35 DEG C, cultivate in 5%CO2 incubators, it is daily aobvious Micro- Microscopic observation cytopathy, culture is terminated after 90% cytopathy occurs in virus control.Its CPE of Microscopic observation.Using Reed-Muench methods calculate medicine medium effective concentration (EC₅₀) (concentration for 50%of maximal effect,EC₅₀).According to the medicine median toxic concentration (TC under 2.2.4₅₀) and medicine medium effective concentration (EC₅₀), calculate The malicious index (TI) of the suppression of medicine.

Formula (3) ... TC₅₀=[Anti log (log is higher than 50% lesion rate drug dilution angle value-pd)] × initial concentration

It is EC50 that cell survival rate, which is more than 50% last concentration,

Formula (4) ... EC₅₀=[Anti log (log is higher than 50% survival rate drug dilution angle value-pd)] × initial concentration

3 experimental results

The result that 3.1 virus virulences are determined

The lesion situation of cell caused by observation virus, the types of HSV- I viral (F plants) are calculated by ReedMuench methods TCID₅₀=10^-3.33The TCID of/100 μ l, RSV Strain₅₀For 10^-2.50The TCID of/100 μ l, EV71 Strain₅₀For 10^-4.33/ 100μl。

Inhibitory action result of 3.2 medicines to virus

As a result it is as shown in Figure 1.By the CPE percentage of record, calculate medicine half using Reed-Muench methods and be poisoned Concentration (TC₅₀) and medicine medium effective concentration (EC₅₀), and determine to press down malicious index (TI=TC₅₀/EC₅₀).It the results are shown in Table 1, Fig. 2.

The selection result of the Echinacea extracorporeal antivirus effect of table 1 spectrum

Note:- representing invalid, TI values are considered as effectively more than 4.

4. discuss

This experiment has investigated effect of the Echinacea opposed polarity position to HSV- I, RSV, EV71 institute cytopathogenic effect simultaneously, Make the result of antiviral spectrum more accurate comprehensive.As a result show, Echinacea difference extract is to thin caused by HSV- I, RSV, EV71 Born of the same parents' lesion has different degrees of inhibitory action.By detecting that Echinacea opposed polarity extract resists to HSV- I, RSV, EV71 Virus effectiveness finds that 80% alcohol reflux, decocting boil position has preferable inhibitory activity to three kinds of viruses.By relatively each more effective TI values find, 80% alcohol reflux position is boiled suppression of the position to HSV, RSV, EV71 to RSV, EV71 inhibition, decocting and imitated Fruit is all higher than the antiviral effect of positive control drug Ribavirin.The purpose of this problem be find Echinacea it is antiviral effectively into Point, and decocting boil position confrontation RSV best results (TI values have reached 196, much larger than positive control 31), therefore selection purple Chrysanthemum water position is bored to RSV effect as tracking foundation, subsequent experimental direction is used as.

Test the optimization of two Echinacea extracting methods

Position is boiled in the decocting of clear and definite Echinacea in experiment one has more preferably to RSV institute's cytopathogenic effects than other polar fractions Inhibition, therefore water extraction on the basis of optimize to determine a more preferable extraction scheme.

Because the main purpose of this research is qualitative, therefore only select to influence extraction effect larger temperature as investigation Factor, sets water boiling and extraction and two kinds of extracting methods of water ultrasonic extraction respectively, and temperature factor is investigated to extracting by experiment in vitro The influence of effect, so that it is determined that optimum extraction scheme.

1 laboratory apparatus and material

1.1 medicine Echinacea dry aerial parts medicinal materials are purchased from Anhui Province Tongcheng City Wei Qing medicinal plants Co., Ltd, powder Broken machine is crushed, standby；Ribavirin injection (Qilu Pharmaceutical Co., Ltd., specification is 50mg/mL, lot number 1501166141).

(VERO, RSV the sensitive cells strain, by Shandong Academy of Medical Sciences microorganism of 1.2 cell African green monkey kidney cells Laboratory is preserved.

1.3 viral seed culture of viruses RSV：Long plants, in October, 2000 quoted from Virology Inst., China Academy of Preventive Medicine Sciences's seed culture of viruses room, This section office preserves.

1.4 reagent distilled water；Cell culture fluid (1640 cell culture fluids, GIBCO products, containing 11% NBCS and 100U/mL penicillin and streptomysin, put 4 DEG C it is standby)；(1640 cell culture fluids, GIBCO products are new containing 2% for cell maintenance medium Raw cow's serum and 100U/mL penicillin and streptomysin)；PBS (pH7.2,8g containing NaCl, KCl 0.2g, KH₂PO₄ 0.2g、 Na₂HPO₄2.9g, plus distilled water, to 1000mL, filtration sterilization, 4 DEG C of separating device is standby)；Cell dissociation buffer (0.25% pancreatin, Macgene Products, dispense to penicillin bottle, put -20 DEG C of refrigerators standby).

1.5 instruments are with experiment one.

2. experimental method

The extraction of 2.1 opposed polarity Echinacea Purpurea Herb P.Es

(1) water ultrasonic extraction takes Echinacea powder 40g, plus 8 times of distilled water ultrasonic extraction 2h measured, suction filtration；Filter residue is again Plus 8 times of amount distilled water repeat to extract, suction filtration；Filtrate merges twice, and 100 DEG C of water-baths are volatilized, and cell maintenance medium dissolving is appropriate, and 0.22 μm filter filtration sterilization, it is standby.

(2) water boiling and extraction method takes Echinacea medicinal powder 40g, and the distilled water for adding 8 times of amounts carries out decoction 2h, suction filtration； The dregs of a decoction plus same amount distilled water after filtering is taken to continue to decoct 2h, suction filtration；Filtrate it will merge twice, water-bath is volatilized.Maintained with cell Liquid dissolving is appropriate, and 0.22 μm of filter filtration sterilization is standby.

2.2 antiviral study in vitro

2.2.1 the measure of drug cytotoxicity

Take Echinacea water sonicated extract, water boiling and extraction thing appropriate, 2 multiple proportions are made of cell maintenance medium and are serially diluted, altogether 12 dilution factors, are loaded onto on the vero cells for having grown up to individual layer in porous plate, and each dilution factor repeats 3 holes, in 37 DEG C, 5%CO₂Cultivated in incubator.Micro- Microscopic observation lesion situation after 24 hours, treats that virus control lesion rate reaches 90%, termination Culture, is observed and records cytopathy (CPE) percentage, and medicine median toxic concentration is calculated using Reed-Muench methods (TC₅₀), and determine maximal non-toxic concentration (TC₀)。

2.2.2 the influence that medicine is acted on pathological changes caused by virus

Take Echinacea water boiling and extraction thing, water sonicated extract to make 2 multiple proportions of cell maintenance medium and be serially diluted, totally 12 it is dilute Degree of releasing, is sequentially added on the individual layer vero cells in 96 orifice plates in (100 μ L/ holes), and each dilution factor repeats 3 holes, if Ribavirin Parenteral solution stoste is positive control, and sets cell controls, virus control.In addition to cell controls plus 100 μ L maintaining liquids, each hole adds 100 TCID50 viral 100 μ L.Put 35 DEG C, 5%CO₂Cultivated in incubator, daily micro- Microscopic observation cytopathy is treated Virus control occurs terminating culture after 90% cytopathy.Microscopic observation simultaneously records its CPE.Using Reed-Muench method meters Calculate medicine medium effective concentration (EC₅₀) (concentration for 50%of maximal effect, EC₅₀).According to upper State medicine median toxic concentration (TC₅₀) and medicine medium effective concentration (EC₅₀), calculate the malicious index (TI) of medicine suppression.

3 experimental results (are shown in Table 2, Fig. 3).

The Echinacea modifiedly extracted method result of table 2

4. discuss

Temperature can influence the dissolution efficiency by thermal lability composition, two in experiment kind extracting method mainly investigate temperature because Influence of the element to active component extraction efficiency.This experimental result shows that the malicious index of suppression of water boiling and extraction is more than water ultrasonic extraction The malicious index of suppression, while the effect of water extract is much larger than the effect of positive control drug Ribavirin.It follows that：Suppress The active ingredient of RSV institutes cytopathogenic effect still has presence in a heated condition, and hot conditions are conducive to its dissolution, it is thus determined that Water boiling and extraction method is final extracting method.

Test the isolation and purification of three Echinacea antivirus effective positions

The application of macroporous absorbent resin appears in nineteen sixties, is the another separation after ion-exchange Purification process, the seventies start isolating and purifying applied to Chinese medicine.Macroporous absorbent resin passes through intermolecular force, hydrogen bond The molecular sieve effect of suction-operated and loose structure plays a role, and material to be separated exists because of the difference of absorption affinity and molecular size range The purpose of isolation and purification is reached in elution process.It is different with selected monomer molecule structure by its polarity size, it can be divided into Nonpolar, middle polarity and polarity three types, preferable separating effect is respectively provided with to the medicine of various opposed polarities.D101 types are big Macroporous adsorbent resin is the nonpolar EVA of the dilute type of benzene second, is a kind of artificial synthesized Polymer adsorption of porous spongy structure Agent, the scope of application compares a wider spectrum.

The water soluble part of Echinacea is mainly the big polar substances such as Caffeic acids derivative, polysaccharide, protein, composition Species is various and polarity is close, and separating difficulty is larger.Existing document report, caffeic acid derivative class is the important stimulation of Echinacea The active component of immunity of organisms, in the case where intending as target components, selects D101 type macroporous absorbent resins, can be by Big polar component is quickly eluted, and accomplishes that active principle loses less, and comparatively ideal purpose is isolated and purified so as to reach.

1 laboratory apparatus and material

Ethanol (Tianjin Fu Yu Fine Chemical Co., Ltd product is analyzed pure)；D101 types macroporous absorbent resin (Cangzhou treasured grace Chemical Co., Ltd.)；Glass chromatography column (4.5 × 85cm of Φ).Remaining instrument is with material with experiment two.

2. experimental method

It is prepared by 2.1 sample solutions

Echinacea powder 50g is taken, the distilled water for adding 8 times of amounts carries out decoction 2h, suction filtration；Take the dregs of a decoction plus same amount after filtering Distilled water continues to decoct 2h, suction filtration；Filtrate it will merge twice, 100 DEG C of water-baths are volatilized.Above-mentioned water boiling and extraction thing ultrasound is taken to be dissolved in It is standby in 400ml water

The pretreatment of 2.2 macroreticular resins

A certain amount of D101 types macroporous absorbent resin is taken, 24h is soaked with 100% ethanol, the volume after being fully swelled is about 1L.The glass chromatography column (4.5 × 85cm of Φ) of cleaning is taken, wet method dress post is about at the 2/3 of pillar height, to use ethanol to resin height It is rinsed.Detection post lower end trickle, i.e., press 1 by liquid and water at any time:5 mixing, if resulting solution clarification is saturating after mixing When bright, distilled water flushing is used instead, the liquid flowed out into post stands resin column bed standby without ethanol flavor.

2.3 loading

Sample solution in 2.1 is splined in the macroporous resin column handled well with the slow drainage of glass bar, layer is opened The valve of post lower end is analysed, allows sample solution slowly to flow, finally keeps liquid level to be higher by 2~3cm of resin column bed, closes valve, Stand 12 hours.

2.4 elution

Successively gradient elution is carried out with water, 25% ethanol, 50% ethanol, 75% ethanol.Control the efflux rate of outflow about For 1 drop/sec, one section is collected as per 200ml, each gradient is rushed to colourless and changes next gradient.Eluent is suitable by collecting Sequence is numbered, and water bath method respectively, standby.

2.5 antiviral study in vitro

2.5.1 the measure of drug cytotoxicity

Take Echinacea macroreticular resin eluate appropriate, dissolved respectively with cell maintenance medium, and do 2 multiple proportions and be serially diluted, altogether 12 dilution factors, are loaded onto in 96 orifice plates and have grown up on the vero cells of individual layer, and each dilution factor longitudinally repeats 3 holes, in 37 DEG C, 5%CO₂Cultivated in incubator.Micro- Microscopic observation lesion situation after 24 hours, treats that the lesion rate of virus control reaches 90%, culture is terminated, observes and records cytopathy (CPE) percentage, is calculated using Reed-Muench methods in medicine half Malicious concentration (TC₅₀)。

2.5.2 the influence that medicine is acted on pathological changes caused by virus

Take Echinacea macroreticular resin eluate to dissolve and make 2 multiple proportions of cell maintenance medium to be serially diluted, totally 12 dilution factors, It is loaded onto successively on the individual layer vero cells in 96 orifice plates in (100 μ L/ holes), each dilution factor repeats 3 holes, if Ribavirin is injected Liquid stoste is positive control, and sets cell controls, virus control.In addition to cell controls plus 100 μ L maintaining liquids, each hole adds 100 TCID₅₀The μ L of RSV virus liquids 100.Put 35 DEG C, 5%CO₂Cultivated in incubator, daily micro- Microscopic observation cytopathy treats disease Poison control occurs terminating culture after 90% cytopathy.Microscopic observation simultaneously records its CPE.Calculated using Reed-Muench methods Medicine medium effective concentration (EC₅₀) (concentration for 50%of maximal effect, EC₅₀).According to above-mentioned Medicine median toxic concentration (TC₅₀) and medicine medium effective concentration (EC₅₀), calculate the malicious index (TI) of medicine suppression.

3 experimental results (are shown in Table 3, Fig. 4)

The antivirus action result of the Echinacea water extract D101 type macroreticular resin eluates of table 3

4. discuss

In elution process, to ensure that composition does not lose, initially outflow, also do not divided in equal volume by macroporous resin adsorption part Section is collected；200ml is set during collection as a collection body, to ensure more preferable separating effect.Eluate serial number in table 3 After Antiviral breeding checking, the result in order merging the close collection section of adjacent ten effects.

Result is shown in chart, and water 2, water 3, water 4, water 5, the malicious index of suppression of water 6 separate pure more than D101 types macroreticular resin The malicious index (TI=235) of suppression before change, and still better than Ribavirin (TI=32), illustrate to be made with D101 macroporous absorbent resins Isolation and purification method for Echinacea water boiling and extraction thing is practical.

Make a general survey of the external anti respiratory syncytial virus result in this part to find, the antiviral effect at basic each elution position is excellent In Ribavirin, there are two kinds of guesses for such case：1. richness of the isolation and purification method that this experiment is used to active component Collect effect undesirable；2. there are a variety of anti respiratory syncytial virus active components in Echinacea, and strongly active composition is mainly concentrated At the water position of big polarity.Which kind of guess no matter this experiment meet, and illustrates, other in addition to the effect preferably gone out concentrates position Still there is the value of continual exploitation at elution position, very important.

Test analysis and the Structural Identification of the antiviral active ingredient of four Echinaceas

1 laboratory apparatus and material

1.1 medicines take three reclaimed waters 2 of experiment, water 3, water 4, water 5, water 6 to merge, 4000rpm centrifugation 15min, supernatant, precipitation 100 DEG C of water-baths are volatilized respectively, standby.

1.2 reagent alcohols, butyl acetate, formic acid, phosphoric acid (Tianjin Fu Yu Fine Chemical Co., Ltd product is analyzed pure)； Acetonitrile (TEDIA company, Yu Wang groups, chromatographically pure)；

1.3 instrument PAD UV-detectors；High performance liquid chromatograph；The series of high efficiency liquid chromatographs of Agilent 1260 (Agilent company of the U.S., equipped with on-line degassing machine, quaternary pump, automatic sampler, column oven, PDAD)； Agilent 6230TOF mass spectrographs (Agilent company of the U.S., equipped with standard electric spray ion source, MassHunler Data Acquisition, which works online, to stand and Qualiatise Analysis off-line analysis software)；Pressed in CHEETAHTMMP100 fast Speed purifying preparation system (Tianjin Bonaaijieer Technology Co., Ltd).Remaining same experiment three.

2. experimental method and result

The selection of 2.1 Detection wavelengths

Take and Supernatant samples are centrifuged in this experiment 1.1 in right amount, with distilled water diluting to close to colourless, through PAD UV-detectors Full wavelength scanner, as a result such as Fig. 5.

As shown in Figure 5, PAD monitoring wave-length coverage is 200~500, a length of 330nm of maximum absorption wave of sample, by it It is used as the reference conditions of application high-efficient liquid phase analysis.

2.2 high performance liquid chromatography detection

2.2.1 sample treatment, which takes, centrifuges Supernatant samples in right amount in this chapter, ultrasound is dissolved in ultra-pure water, 0.45 μm of miillpore filter Filtration, obtains test sample, standby.

2.2.2 chromatographic condition

Consulting literatures simultaneously grope by Binding experiment, and the HPLC testing conditions for determining sample are：Syncronis C18 chromatographic columns (250mm×4.6mm,5μm)；35 DEG C of column temperature；Flow velocity 1ml/min；Detection wavelength 330nm；The μ l of sample size 20；Mobile phase condition second The phosphoric acid solution (B)=10 (A) of nitrile (A) -0.3%：90 (B), isocratic elution.

2.2.3 HPLC testing results

It will be appreciated from fig. 6 that the separating resulting composition letter under 2.2.2 chromatographic conditions of the separating obtained sample of D101 macroporous absorbent resins Single, separating degree is good, therefore it is refined that in half preparation solution this sample is mutually continued into purifying.

Combiflash companion condition is pressed in 2.3

This experiment uses SQ230020-0 Flash spherical AQC18 (spherical 20~35 μm, 20g) chromatographic column, fast in middle pressure Speed is prepared in liquid phase, and mobile phase is used as using the phosphoric acid of acetonitrile -0.3%；Flow velocity：15ml/min；Column temperature：Room temperature；Collect wavelength： 325nm。

Table 4 quickly prepares the gradient of liquid phase

The preparation of 2.4 compounds

Weigh the Supernatant samples 0.0972g in 1.1, the ultrasonic dissolution in 1ml distilled water, sample introduction.By quick under 2.3 Chromatographic condition preparation is prepared, product is prepared by peak collection, and number.

Product will be prepared and cross 0.45 μm of miillpore filter respectively, by the chromatographic condition under 2.2.2, sample introduction high performance liquid chromatography Detected, obtain unimodal material, see Fig. 7.

According to high performance liquid chromatography detection result, merge spectrogram same composition, 40 DEG C of waters bath with thermostatic control are volatilized, and obtain white needles Crystallization.The Structural Identification of 2.5 compounds

Cichoric acid reference substance：ESI-MS:492.1135[M+H₂O]⁺, molecular weight 374.1, molecular formula is C₂₂H₁₈O₁₂.Cracking Fragment when voltage is 100V：135.0431,163.0384,224.1249,295.0446,325.0710,391.2831, 492.1139,619.1109,767.1069,841.0523,922.0092,966.1917,1109.1847；Cracking voltage is Cleaved fragment during 200V has：135.0441,163.0390,295.0454,378.9861,492.1132,569.0255, 691.0268,767.1098,841.0469,922.0094,966.1924,1109.1796.As shown in Figure 8.

Under same experimental conditions, the Dissociation fragment of gained sterling compound：When cracking voltage for 100V, fragment has： 163.0389,224.1273,295.0450,325.0694,391.2831,457.0759,492.1135,619.1061, 731.1020,841.0503,922.0093,966.1908,1111.1960；When cracking voltage for 200V, fragment has： 135.0441,163.0392,295.0469,378.9877,492.1126,619.1096,731.1036,841.0484, 922.0099,966.1929,1111.1909.As shown in Figure 9.Dissociation fragment with Cichoric acid reference substance is basically identical, therefore can sentence Resulting white, needle-shaped crystals of breaking are Cichoric acid.

The pharmacy in vitro experiment of 3 sterling compounds

The measure of 3.1 drug cytotoxicities

Take preparation sterling, dissolved with cell maintenance medium, cross 0.45 μm of miillpore filter, do 2 multiple proportions and be serially diluted, totally 12 it is dilute Degree of releasing, is loaded onto on the vero cells for having grown up to individual layer in porous plate, and each dilution factor repeats 3 holes, in 37 DEG C, 5%CO₂Training Support in case and cultivate.Micro- Microscopic observation lesion situation after 24 hours, treats that the lesion rate of virus control reaches 90%, terminates culture, Observe and record cytopathy (CPE) percentage, medicine median toxic concentration (TC is calculated using Reed-Muench methods₅₀)。 The influence that 3.2 medicines are acted on pathological changes caused by virus

Take the Supernatant samples in 1.1 and prepare sterling in right amount, dissolved with cell maintenance medium, cross 0.45 μm of miillpore filter, used Cell maintenance medium, which dissolves and does 2 multiple proportions, to be serially diluted, and totally 12 dilution factors, are loaded onto the individual layer vero cells in 96 orifice plates successively Upper (100 μ L/ holes), each dilution factor repeats 3 holes, if ribavirin injection stoste is positive control, and sets cell controls, disease Poison control.In addition to cell controls plus 100 μ L maintaining liquids, each hole adds 100 TCID₅₀The μ L of RSV virus liquids 100.Put 35 DEG C, 5% Cultivated in CO2 incubators, daily micro- Microscopic observation cytopathy terminates training after 90% cytopathy occurs in virus control Support.Microscopic observation simultaneously records its CPE.Medicine medium effective concentration (EC is calculated using Reed-Muench methods₅₀) (concentration for 50%of maximal effect, EC₅₀).According to said medicine median toxic concentration (TC₅₀) And medicine medium effective concentration (EC₅₀), calculate the malicious index (TI) of medicine suppression.

3.3 experimental results (as shown in table 5, Figure 10)

The Echinacea of table 5 prepares sterling Antiviral breeding result

4. discuss

Gained compound Cichoric acid is prepared in this experiment is to the inhibition index of Respiratory Syncytial Virus(RSV) institute cytopathogenic effect 4272, it is 9 times of supernatant before preparing, is 256 times of positive control drug, suppression toxic effect fruit is prominent, therefore is external preventing respiratory The active ingredient of syncytial virus.

Fig. 4 in experiment three, water 2 that antiviral study in vitro preferably goes out, water 3, water 4, water 5, the position of water 6 it is disease-resistant Toxic effect fruit difference is larger, because each position sample size is less, to be smoothed out subsequent experimental, the sample used in this experiment For the consolidated material at each position.

Due to still there is the macromolecular substances such as polysaccharide, protein, tannin in macroreticular resin eluate, after this experiment is using centrifugation The mode of supernatant is taken to remove big molecular impurity, to reduce purifying difficulty.

Sample finds that it is the 4th peak in gross sample that compound, which is made, with preparing the HPLC collection of illustrative plates of product before prepared by contrast, Its content highest in gross sample, therefore be easier to get.

Summarize

This research is supplemented and the perfect character of three kinds of separate sources Echinaceas, microscopical characters data；Establish Echinacea Extracorporeal antivirus effect spectrum；Followed the trail of by external pharmacological activity and be found that active component Cichoric acid.The Experiment Result of this research is purple Accurate use for boring the antiviral further research of chrysanthemum and three kinds of separate sources Echinaceas provides science data and data.

This experiment in the screening operation of antiviral activity composition, take " screening scope by width to it is narrow, from the superficial to the deep, The experimental program of progressively lock onto target ".The different type virus with different inhereditary materials is selected first, with reference to opposed polarity portion Position extract does wide spectrum screening, and comprehensive analysis the selection result determines best results polar fraction and viral species, and then with effective It is the separation at position, purifying, refined as tracking direction, using selected viral pharmacy in vitro as screening foundation, promote, enter layer by layer And obtain antiviral active substance.

Current to trigger the treatment of disease to have no specific drug for Respiratory Syncytial Virus(RSV), Ribavirin is clinical conventional Medicine.In this experiment, the positive control drug that Ribavirin is Antiviral breeding is selected, although its extracorporeal extracorporeal suppression is not protruded, But can not negate its status in clinical practice.For ensure experimental result uniformity, should continue using Ribavirin as The positive control drug for investigating Cichoric acid antiviral effect is applied to internal pharmacological evaluation, also it is contemplated that increasing other positive controls Medicine.

Cichoric acid in Echinacea is labile element, and content is unstable in the preparation commodity of Echinacea or does not deposit for it , therefore often it is used as quality standard using polysaccharide constituents or acid amides constituents in quality control.The display of this experimental result, Cichoric acid The external RSV effects that suppress are protruded, and show that its pharmacological activity is affirmed, thus its content in curative drug can be to certain The curative effect of a little diseases can produce certain influence.Therefore, one is entered in follow-up internal pharmacological evaluation, in Echinacea prescribed preparation In step research, for the effective utilization and its action effect of Cichoric acid, further investigation can be also done.

Echinacea is used in the therapeutic process of human viral disease realizing, still has medicinal material shortage of standard, disease-resistant Cytotoxic activity composition is unknown, antivirus action mechanism, lie across at the moment the problems such as target spot is unclear.Therefore pharmacological activity is carried out to Echinacea It is the premise that it obtains further development and application in China to study, find reliable active component and carry out total quality evaluation.

Claims (1)

1. application of the Cichoric acid in the medicine of anti respiratory syncytial virus is prepared.